It is generally believed that multiple Ca2+ stores are present in cells, a notion that has now been made substantive by the discovery of multiple Ca2+ mobilizing messengers. Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two such messengers that are derived from NAD and NADP, respectively. A wide variety of cells, from plants to mammals, including human, have been shown to be responsive to these two novel Ca2+ messengers. Not only are their structures and mechanisms of action different, their targeted Ca2+ stores are also distinct and separable. Moreover, they are derived from different substrates, cADPR from NAD and NAADP from NADP. It is thus entirely unexpected that a single enzyme is responsible for synthesizing these two different Ca2+-messengers. CD38 is one such enzyme and it belongs to a new family of homologues that includes CD157 and Aplysia ADP-ribosyl cyclase. Results using knockout mice show that CD38 is the most dominant enzyme in mammalian tissues responsible synthesizing cADPR and NAADP. The emerging view is that cADPR and NAADP represent two branches of a novel Ca2+-signaling pathway with CD38 at its bifurcation point. Because of its pivotal role in the cADPR/NAADP-signaling pathway, it is of critical importance that the molecular mechanism of its action be elucidated. We have expressed the extra-membrane domain of CD38 in yeast as a soluble protein that retains all enzymatic activities indistinguishable from native CD38. We have mapped the active site and identified the critical residues in it by site-directed mutagenesis. These results form the basis for a unified catalytic model that accounts for the novel multiplicity of this family of signaling enzymes. We have now succeeded in crystallizing CD38 and solved its structure. The structure-function studies described in this proposal will verify and refine the catalytic model and the specific aims are: Aim 1: To determine the structural basis of the multi-catalytic functions of CD38. Aim 2: To determine the structural basis of regulation of CD38 catalysis. Aim 3: To determine the structural basis of the antigenic functions of CD38. Aim 4: To characterize intracellular CD38.